【摘要】 目的构建携带人生存素(survivin)基因短发夹RNA(shRNA)表达载体的重组腺病毒。方法构建并筛选重组质粒pShuttles-urvivin,将其与腺病毒骨架质粒共转染HEK-293细胞,经同源重组产生腺病毒;PCR鉴定并测定病毒滴度;β-半乳糖染色检测病毒对人乳腺癌细胞MCF-7的感染效率;流式细胞术、RT-PCR和W estern免疫印迹法检测其生物学功能。结果经PCR鉴定重组腺病毒构建成功;48 h后感染率达75%以上;腺病毒感染MCF-7细胞后48 h,生存素mRNA和蛋白的表达均受到抑制;腺病毒感染MCF-7细胞后,细胞分裂受阻于G2/M期,在48和72 h有凋亡峰出现。结论成功构建人生存素基因shRNA表达载体的重组腺病毒。更多还原

【Abstract】 AIM To construct recombinant adenovirus with short hairpin RNA(shRNA) expression vector of survivin.METHODS After pShuttle-survivin was constructed and screened,cotransfected it with bone plasmid of adenovirus into HEK-293 cells to get recombinant adenovirus.Virus titer was determined after verification by PCR.Efficiency of adenovirus infecting MCF-7 cells was detected by β-galactose staining.Its biological effects were observed by means of flow cytometric analysis,RT-PCR and Western blot.RESULTS Adenovirus was constructed successfully.Total of 75% of MCF-7 cells were infected after treated with adenovirus.After infected with adenovirus,RT-PCR and Western blot analysis showed that survivin was suppressed at 48 h on the level of mRNA and protein.Flow cytometric analysis indicated that survivin shRNA induced the cell cycle arrest at G2/M phase,and induced apoptosis at 48 and 72 h.CONCLUSION Recombinant adenovirus with shRNA targeting survivin may be an effective way of gene therapy on human breast cancer.更多还原